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United States Patent |
5,741,804
|
Keenan
,   et al.
|
April 21, 1998
|
Substituted benzimazoles which inhibit platelet aggrecation
Abstract
This invention relates to compounds of the formula:
##STR1##
which are effective for inhibiting platelet aggregation, pharmaceutical
compositions for effecting such activity, and a method for inhibiting
platelet aggregation.
Inventors:
|
Keenan; Richard McCulloch (Malvern, PA);
Miller; William Henry (Schwenksville, PA)
|
Assignee:
|
SmithKline Beecham Corporation (Philadelphia, PA)
|
Appl. No.:
|
446791 |
Filed:
|
May 30, 1995 |
PCT Filed:
|
December 1, 1993
|
PCT NO:
|
PCT/US93/11779
|
371 Date:
|
May 30, 1995
|
102(e) Date:
|
May 30, 1995
|
PCT PUB.NO.:
|
WO94/12478 |
PCT PUB. Date:
|
June 9, 1995 |
Foreign Application Priority Data
Current U.S. Class: |
514/394; 514/322; 514/388; 514/419; 546/199; 548/308.7; 548/310.1; 548/494 |
Intern'l Class: |
C07D 235/12; A61K 031/415 |
Field of Search: |
548/308.7,310.1
514/388,394
|
References Cited
U.S. Patent Documents
4634783 | Jan., 1987 | Fujii et al. | 549/475.
|
5272167 | Dec., 1993 | Girijavallabhan et al. | 514/394.
|
Primary Examiner: Richter; Johann
Assistant Examiner: Stockton; Laura L.
Attorney, Agent or Firm: McCarthy; Mary E., Venetianer; Stephen, Lentz; Edward T.
Claims
What is claimed is:
1. A compound of the formula (I):
##STR13##
wherein: R is R.sup.7, --CH.sub.2 R.sup.7, --CH.sub.2 CH.sub.2 R.sup.7,
--CH.sub.2 CH.sub.2 CH.sub.2 R.sup.7, --NHCH.sub.2 R.sup.7, or
--NHCH.sub.2 CH.sub.2 R.sup.7 ;
R* is H or --(CH.sub.2).sub.1-2 phenyl;
R.sup.6 is W--(CR'.sub.2).sub.q --Z--(CR'R.sup.10).sub.r
--U--(CR'.sub.2).sub.s --; (CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s is
CH.sub.2 NHCO, CH(NR'R")CONH, CONH or NHCO;
R.sup.7 is --CO.sub.2 H;
##STR14##
or R"HNCO--;
each R' independently is H, C.sub. 1-4alkyl, C.sub.3-7 cycloalkyl-C.sub.0-4
alkyl, or Ar--C.sub.0-4 alkyl;
each R" independently is R' or --C(O)R';
Z is phenyl; and
q is0to 3;
or a pharmaceutically acceptable salt thereof.
2. A compound which is
›5-(4-aminoiminomethyl)-benzoylamino!benzimidazole-2-propanoic acid or a
pharmaceutically acceptable salt thereof.
3. A compound which is:
›5-(4-(aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-acetic acid;
›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-propanoic acid;
›5-(4-aminomethyl)benzoylamino!benzimidazole-2-amino-N-acetic acid;
›5-(4-aminoiminomethyl)phenylaminocarbonyl!benzimidazole-2-amino-N-acetic
acid;
1-N-benzyl-›6-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
acid;
1-N-benzyl-›5-(4-aminoiminomethylbenzoylamino!benzimidazole-2-propanoic
acid; or
1-N-phenethyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
acid;
or a pharmaceutically acceptable salt thereof.
4. A pharmaceutical composition comprising a compound according to claim 1
and a pharmaceutically acceptable carrier.
5. A pharmaceutical composition comprising a compound according to claim 2
and a pharmaceutically acceptable carrier.
6. A pharmaceutical composition comprising a compound according to claim 3
and a pharmaceutically acceptable carrier.
7. A method for effecting inhibition of platelet aggregation to a subject
in need thereof which comprises administering a compound according to
claim 1.
8. A method according to claim 7 for treating stroke or transient ischemia
attacks or myocardial infarction.
Description
This application is a 371 of PCT/US93/11779 filed Dec. 1, 1993.
FIELD OF THE INVENTION
This invention relates to novel bicyclic compounds which inhibit platelet
aggregation, pharmaceutical compositions containing the compounds and
methods of using the compounds.
BACKGROUND OF THE INVENTION
Platelet aggregation is believed to be mediated primarily through the
fibrinogen receptor, or GPIIb-IIIa platelet receptor complex, which is a
member of a family of adhesion receptors referred to as integrins. It has
been found that frequently the natural ligands of integrin receptors are
proteins which contain an Arg--Gly--Asp sequence (RGD in single letter
amino acid code). Von Willebrand factor and fibrinogen, which are
considered to be natural ligands for the GPIIb-IIIa receptor, possess an
RGD sequence in their primary structure. Functionally, these proteins are
able to bind and crosslink GPIIb-IIIa receptors on adjacent platelets and
thereby effect aggregation of platelets.
Fibronectin, vitronectin and thrombospondin are RGD-containing proteins
which have also been demonstrated to bind to GPIIb-IIIa. Fibronectin is
found in plasma and as a structural protein in the intracellular matrix.
Binding between the structural proteins and GPIIb-IIIa may function to
cause platelets to adhere to damaged vessel walls.
Linear and cyclic peptides which bind to vitronectin and contain an RGD
sequence are disclosed in WO 89/05150 (PCT US88/04403). EP 0 275 748
discloses linear tetra- to hexapeptides and cyclic hexa- to octapeptides
which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation.
Other linear and cyclic peptides, the disclosure of which are incorporated
herein by reference, are reported in EP-A 0 341 915. However, the peptide
like structures of such inhibitors often pose problems, such as in drug
delivery, metabolic stability and selectivity. Inhibitors of the
fibrinogen receptor which are not constructed of natural amino acid
sequences are disclosed in EP-A 0 372A86, EP-A 0 381 033 and EP-A 0 478
363. WO 92/07568 (PCT/US91/08166) discloses fibrinogen receptor antagonism
which mimic a conformational .gamma.-turn in the RGD sequence by forming a
monocyclic seven-membered ting structure. There remains a need, however,
for novel fibrinogen receptor antagonists (e.g. inhibitors of the
GPIIb-IIIa protein) which have potent in vivo and in vitro effects and
lack the peptide backbone structure of amino acid sequences.
The present invention discloses novel bicyclic compounds, including
benzimidazoles. These compounds inhibit binding to the GPIIb-IIIa receptor
and inhibit platelet aggregation.
SUMMARY OF THE INVENTION
In one aspect this invention is a bicyclic compound comprising a
substituted six-membered ring fused to a substituted five-membered ring as
described hereinafter in formula (I).
This invention is also a pharmaceutical composition for inhibiting platelet
aggregation or clot formation, which comprises a compound of formula (I)
and a pharmaceutically acceptable carrier.
This invention is further a method for inhibiting platelet aggregation in a
mammal in need thereof, which comprises internally administering an
effective amount of a compound of formula (I).
In another aspect, this invention provides a method for inhibiting
reocclusion of an artery or vein in a mammal following fibrinolytic
therapy, which comprises internally administering an effective amount of a
fibrinolytic agent and a compound of formula (I). This invention is also a
method for treating stroke, transient ischemia attacks, myocardial
infarction, or atherosclerosis.
DETAILED DESCRIPTION OF THE INVENTION
This invention discloses novel bicyclic compounds which inhibit platelet
aggregation. The novel bicyclic compounds comprise a five-membered ring
fused to an aromatic six membered ring and having a nitrogen-containing
substituent on the six membered ring and an aliphatic substituent,
preferably containing an acidic moiety, on the five membered ring. The
five membered ring may contain heteroatoms, such as nitrogen, oxygen and
sulfur, and the six membered ring may be carbocyclic or contain up to two
nitrogen atoms. The fused 6-5 ring system is believed to interact
favorably with the GPIIb-IIIa receptor and to orient the substituent
sidechains on the six and five membered rings so that they may also
interact favorably with the receptor.
Although not intending to be bound to any specific mechanism of action,
these compounds are believed to inhibit the binding of fibrinogen to the
platelet-bound fibrinogen receptor GPIIb-IIIa, and may interact with other
adhesion proteins via antagonism of a putative RGD binding site.
The compounds of this invention are compounds of formula (I):
##STR2##
wherein: A.sup.1 to A.sup.3 form any accessible substituted five-membered
ring, which may be saturated or unsaturated, optionally containing up to
two heteroatoms chosen from the group of O, S and N wherein S and N may be
optionally oxidized;
D.sup.1 to D.sup.4 form any accessible substituted six membered ring,
optionally containing up to two nitrogen atoms;
R is at least one substituent chosen from the group of R.sup.7, or
Q--C.sub.1-4 alkyl, Q--C.sub.2-4 alkenyl, Q--C.sub.2-4 alkynyl,
Q--C.sub.3-4 oxoalkenyl or Q--C.sub.3-4 oxoalkynyl, Q--C.sub.1-4
aminoalkyl, Q--C.sub.3-4 aminoalkenyl, or Q--C.sub.3-4 aminoalkynyl,
optionally substituted by any accessible combination of one or more of
R.sup.11 or R.sup.7 ;
R* is absent or present as H, Q--C.sub.1-6 alkyl, Q--C.sub.1-6 alkyl,
Q--C.sub.1-6 oxoalkyl, Q--C.sub.2-7 alkenyl, Q--C.sub.3-4 oxoalkenyl,
Q--C.sub.3-4 oxoalkylnyl, Q--C.sub.2-4 alkynyl, C.sub.3-6 cycloalkyl, Ar,
or Het, optionally substituted by one or more of R.sup.11 ;
each Q independently is H, C.sub.3-6 cycloalkyl, Het or Ar;
R.sup.6 is W--(CR'.sub.2).sub.q --Z--(Cr'R.sup.10).sub.r
--U--(CR'.sub.2).sub.S --V--;
each R.sup.7 independently is --COR.sup.8, --COCR'.sub.2 R.sup.9,
--C(S)R.sup.8, --S(O).sub.m OR', --S(O).sub.m NR'R", --PO(OR'),
--PO(OR').sub.2, --B(OR').sub.2, --NO.sub.2, or tetrazolyl;
each R.sup.8 independently is --OR', --NR'R", --NR'SO.sub.2 R', --NR'OR',
--OCR'.sub.2 CO(O)R', or AA;
R.sup.9 is --OR', --CN, --S(O).sub.r R', --S(O).sub.m NR'.sub.2, --C(O)R',
C(O)NR'.sub.2, or --CO.sub.2 R';
R.sup.10 is H, C.sub.1-4 alkyl or --NR'R";
each R.sup.11 independently is H, halo, --OR.sup.12, --CN, --NR'R.sup.12,
--NO.sub.2, --CF.sub.3, CF.sub.3 S(O).sub.r --, --CO.sub.2 R',
--CONR'.sub.2, Q--C.sub.0-6 alkyl--, Q--C.sub.0-6 alkylamino- or
Q--C.sub.0-6 alkyl-S(O).sub.r --; Q--C.sub.2-6 alkynyl--, Q--C.sub.0-6
alkyloxy--, Q--C.sub.0-6 alkylamino- or Q--C.sub.0-6 alkyl-S(O).sub.r --;
each R.sup.12 independently is ', --C(O)R', --C(O)NR'.sub.2, --C(O)OR',
--S(O).sub.m R', or --S(O).sub.m NR'.sub.2 ;
each R' independently is H, C.sub.1-4 alkyl, C.sub.3-7 cycloalkyl-C.sub.0-4
alkyl, or Ar--C.sub.0-4 alkyl;
each R" independently is R' or --C(O)R';
AA is an amino acid with the carboxyl group optionally protected;
U and V are absent or CO, CR'.sub.2, C(.dbd.CR'.sub.2), S(O).sub.n, O, NR',
CR'OR', CR'(OR")CR'.sub.2, CR'.sub.2 CR'(OR"), C(O)CR'.sub.2, CR'.sub.2
C(O), CONR', NR'CO, OC(O), C(O)O, C(S)O, OC(S), C(S)NR', NR'C(S),
S(O).sub.n NR', NR'S(O).sub.n, N.dbd.N, NR'NR', NR'CR'.sub.2,
NR'CR'.sub.2, CR'.sub.2 O, OCR'.sub.2, C.tbd.C, or CR'.dbd.CR', provided
that U and V are not simultaneously absent;
W is R'R"N--, R'R"NR'N--, R'R"NR'NCO--, R'.sub.2 NR'NC(.dbd.NR')--,
##STR3##
or E; X is absent, N.dbd.CR', C(O), or O;
Y is absent, S, or O;
Z is (CH.sub.2).sub.t, Het, Ar, or C.sub.3-7 cycloalkyl;
each m independently is 1 or 2;
each n independently is 0 to 3;
q is 0 to 3;
each r independently is 0 to 2;
each s independently is 0 to 2; and
each t independently is 0 to 2;
or a pharmaceutically acceptable salt thereof.
Also included in this invention are complexes or prodrugs of the compounds
of this invention. Prodrugs are considered to be any covalently bonded
carriers which release the active parent drug according to formula (I) in
vivo.
In cases wherein the compounds of this invention may have one or more
chiral centers, unless specified, this invention includes each unique
nonracemic compound which may be synthesized and resolved by conventional
techniques. In cases in which compounds have unsaturated carbon-carbon
double bonds, both the cis (Z) and trans (E) isomers are within the scope
of this invention. In cases wherein compounds may exist in tautomeric
forms, such as keto-enol tautomers, such as
##STR4##
and tautomers of guanidine-type groups, such as
##STR5##
each tautomeric form is contemplated as being included within this
invention whether existing in equilibrium or locked in one form by
appropriate substitution with R'. The meaning of any substituent at any
one occurrence is independent of its meaning, or any other substituent's
meaning, at any other occurrence, unless specified otherwise.
With reference to formula (I), suitably,
A.sup.1 is CR.sup.1 R.sup.1', CR.sup.1, NR.sup.1, N, O, or S(O).sub.x ;
A.sup.2 is CR.sup.2 R.sup.2', CR.sup.2, or NR.sup.2 ;
A.sup.3 is CR.sup.3 R.sup.3', CR.sup.3, NR.sup.3, N, O, or S(O).sub.x ;
D.sup.1 -D.sup.4 and CR.sup.11, CR.sup.6 or N;
R is (CR.sup.14 R.sup.15).sub.u --(T).sub.v --(CR.sup.14 R.sup.15).sub.w
--R.sup.7 wherein T is CR.sup.14 R.sup.15 --CR.sup.14 R.sup.15,
CR'.dbd.CR' C.tbd.C, or NR' and R.sup.14 and R.sup.15 are R', OR', or
NR'R" provided that R.sup.14 and R.sup.15 are not simultaneously OR' or
NR'R' when they are attached to the same carbon;
R.sup.1 -R.sup.3 and R.sup.1' -R.sup.3' are R* or R;
R.sup.6 is W--(CR'.sub.2).sub.q --Z--(CR'R.sup.10).sub.r
--U--(CR'.sub.2).sub.s ;
x is 0 to 2; and
u, v and w independently are 0, 1, or 2.
More suitably,
A.sup.1 is CR.sup.1 R.sup.1', CR.sup.1, NR.sup.1, N, O or S;
A.sup.2 is CR.sup.2 R.sup.2', NR.sup.2 or CR.sup.2 ;
A.sup.3 is CR.sup.3 R.sup.3', NR.sup.3, N, O, or S;
D.sup.1 and D.sup.4 are CH;
D.sup.2 or D.sup.3 is CR.sup.6 ;
R.sup.2 is R;
R.sup.3 or R.sup.3' is R*; and
R.sup.6 is W--(CR'.sup.2).sub.q --Z--(CR'R.sup.10).sub.r --U.
Suitably, (CR'R.sup.10).sub.r --U--(CR'.sup.2).sub.s --V is CONR', NR'CO,
CH(NR'R")CONH, CH.sub.2 CONH, CONR'CH.sub.2, CONHCH.sub.2, CH.sub.2 CHOH,
CHOHCH.sub.2, CONHCHR'CH.sub.2, CH.sub.2 NHCO.sub.2 CH.sub.2, CH.sub.2
CH.sub.2 NHCO.sub.2, CONHCH.sub.2 CO, CONHCH.sub.2 CHOH, CH.dbd.CHCONH,
NHCO.sub.2 CH.dbd.CH, SO.sub.2 NR'CHR'CH.sub.2, or CH.dbd.CH, or CH.sub.2
CH.sub.2.
Representative compounds of this invention are given by each of formulae
(II)-(VIII):
##STR6##
Preferably, A.sup.1 is CH.sub.2, CH, NR", N, O, or S; A.sup.2 is CR.sup.2
or CR.sup.2 R.sup.2' ; and A.sup.3 is CR.sup.3 R.sup.3, CR.sup.3,
NR.sup.3', N, O, or S.
Preferably, R.sup.1 and/or R.sup.1' are absent or present as H.
Preferably, R.sup.2 is R.sup.7, --CH.sub.2 R.sup.7, --CH.sub.2 CH.sub.2
R.sup.7, --CH.sub.2 CH.sub.2 CH.sub.2 R.sup.7, --NHCH.sub.2 R.sup.7, or
--NHCH.sub.2 CH.sub.2 R.sup.7. Most preferably, R.sup.7 is CO.sub.2 H.
Preferably, R.sup.3 is absent or present as H or --C.sub.1-6 alkyl. Most
preferably, Q is Ar.
Preferably, (CR'R.sup.10).sub.r --U--(CR'.sub.2)s is CONH or NHCO.
Preferably, Z is phenyl.
Preferably, W is R'R"N--, R'R"NC(.dbd.NR'), or R'R"NC(.dbd.NR')NR'--,
R'R"NCO--, or N wherein R' and R" are preferably H.
Most preferably, R.sup.6 is
##STR7##
In one group of preferred compounds, R.sup.1 is absent; R.sup.2 is CH.sub.2
CO.sub.2 H, CH.sub.2 CH.sub.2 CO.sub.2 H, CH.sub.2 CH.sub.2 CH.sub.2
CO.sub.2 H, NHCH.sub.2 CO.sub.2 .sup.H, or NHCH.sub.2 CO.sub.2 H; R.sup.3
is H, CH.sub.2 --Ar, or CH.sub.2 CH.sub.2 --Ar; Z is phenyl; W is H.sub.2
N--, H.sub.2 NC(.dbd.NH)--, H.sub.2 NCO--, or H.sub.2 NC(.dbd.NH)NH--; and
(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s --V is CH.sub.2 NHCO,
CH(NR'R")CONH, CONH, N(CH.sub.3)CO or NHCO.
Particular compounds of the invention include, but are not limited to, the
following:
›5-(4-(aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-acetic acid;
›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-propanoic acid;
›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic acid;
›5-(4-aminomethyl)benzoylamino!benzimidazole-2-amino-N-acetic acid;
›5-(4-aminoiminomethyl)phenylaminocarbonyl!benzimidazole-2-amino-N-acetic
acid;
1-N-benzyl-›6-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
acid;
1-N-benzyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
acid; and
1-N-phenethyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
acid;
or a pharmaceutically acceptable salt thereof.
The most preferred compound of this invention is
›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic acid or a
pharmaceutically acceptable salt thereof.
In the above description of formula (I), preferably only one of A.sup.1 to
A.sup.3 are substituted by R, and only one of D.sup.1 -D.sup.4 is
substituted by R.sup.6. W represents a nitrogen-containing group which is
capable of making a hydrogen bond: Preferably W is a basic nitrogen
moiety. R.sup.7 represents a group with a non-bonding pair of electrons
which is capable of forming a hydrogen bond or chelating with a metal.
Preferably R.sup.7 is acidic. It is also preferred that 10-15 intervening
covalent bonds via the shortest intramolecular path will exist between the
group R.sup.7 and W for optimal spacing between these groups, and the
moieties T, U, V and Z, and the alkyl spacers represented by q, r, s, u, v
and w are chosen accordingly.
Abbreviations and symbols commonly used in the peptide and chemical arts
are used herein to describe the compounds of this invention. In general,
the amino acid abbreviations follow the IUPAC-IUB Joint Commission on
Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984).
Arg refers to arginine, MeArg refers to N.sup..alpha. -methyl-arginine,
HArg refers to homoarginine, NArg refers to norarginine, (Me2)Arg refers
to N',N"-dimethyl arginine, (Et.sub.2)Arg refers to N',N"-diethyl arginine
and Orn refers to ornithine. These radicals are suitable components of the
substituent R.sup.6. N.sup..alpha. -Substituted derivatives of these amino
acid are also useful in this invention. Representative methods for
preparing co-substituted derivatives are disclosed in U.S. Pat. No.
4,687,758; Cheung et al., Can. J. Chem., 55, 906 (1977); Freidinger et
al., J. Org. Chem., 48, 77, (1982); and Shuman et al., PEPTIDES:
PROCEEDINGS OF THE 7TH AMERICAN PEPTIDE SYMPOSIUM, Rich, D., Gross, E.,
Eds, Pierce Chemical Co., Rockford, Ill. ,617 ( 1981 ), which are
incorporated herein by reference.
C.sub.1-4 alkyl as applied herein means carbon chains which are branched or
unbranched and includes methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl and t-butyl. C.sub.1-6 alkyl additionally includes pentyl,
n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers
thereof. C.sub.0-4 alkyl and C.sub.0-6 alkyl additionally indicates that
no alkyl group need be present (e.g. that a covalent bond is present).
C.sub.2-6 alkenyl as applied herein means an alkyl group of 2 to 6 carbons
wherein a carbon-carbon single bond is replaced by a carbon-carbon double
bond.
C.sub.2-6 alkenyl includes ethylene, 1-propene, 2-propene, 1-butene,
2-butene, isobutene and the several isomeric pentenes and hexenes. Both
cis and trans isomers are included.
C.sub.2-6 alkynyl means an alkyl group of 2 to 6 carbons wherein one
carbon-carbon single bond is replaced by a carbon-carbon triple bond.
C.sub.2-6 alkynyl includes acetylene, 1-propyne, 2-propyne, 1-butyne,
2-butyne, 3-butyne and the simple isomers of pentyne and hexyne.
C.sub.1-4 oxoalkyl refers to an alkyl group of up to four carbons wherein a
CH.sub.2 group is replaced by a C(O), or carbonyl, group. Substituted
formyl, acetyl, 1-propanal, 2-propanone, 3-propanal, 2-butanone,
3-butanone, 1- and 4-butanal groups are representative. Cl-6oxoalkyl
includes additionally the higher analogues and isomers of five and six
carbons substituted by a carbonyl group. C.sub.3-6 oxoalkenyl and
C.sub.3-6 oxoalkynyl refers to a C.sub.3-6 alkenyl or C.sub.3-6 alkynyl
group wherein a CH.sub.2 group is replaced by C(O) group. C.sub.3-4
oxoalkenyl includes 1-oxo-2-propenyl, 3-oxo-1-propenyl, 2-oxo-3-butenyl
and the like.
C.sub.1-4 aminoalkyl as applied herein means an alkyl group of 1 to 4
carbon atoms that is attached to an amino group. C.sub.1-4 aminoalkyl
includes aminomethyl, aminoethyl, aminopropyl, and aminobutyl. C.sub.3-4
alkenyl and C.sub.3-4 aminoalkynyl refer to a C.sub.3-4 alkynyl or
C.sub.3-4 alkenyl group that is attached to an amino group. Q--C.sub.1-4
aminoalkyl group and the like refer to a C.sub.1-4 aminoalkyl group
wherein in any position a carbon-hydrogen bond is replaced by a carbon-Q
bond.
A substituent on a C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.1-4 aminoalkyl, or C.sub.1-6 oxoalkyl group, such as R.sup.11 or
R.sup.7, may be on any atom which results in a stable structure, and is
available by conventional synthetic techniques.
Q--C.sub.1-6 alkyl refers to a C.sub.1-6 alkyl group wherein in any
position a carbon-hydrogen bond is replaced by a carbon-Q bond.
Q--C.sub.2-6 alkenyl and Q--C.sub.2-6 alkynyl have a similar meaning with
respect to C.sub.2-6 alkenyl and C.sub.2-6 alkynyl.
Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or
naphthyl substituted by one to three moieties R.sup.11. In particular,
R.sup.11 may be C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 alkthio,
trifluoroalkyl, OH, Cl, Br or I.
Het, or heteroaryl, indicates an optionally substituted five or six
membered aromatic ring, or a nine or ten-membered aromatic ring containing
one to three heteroatoms chosen from the group of nitrogen, oxygen and
sulfur, which are stable and available by conventional chemical synthesis.
Illustrative heterocycles are imidazole, benzimidazole, pyrrole, indole,
pyridinyl, quinoline, benzofuryl, furyl, benzopyranyl, benzothiophene or
thiophene. Any accessible combination of up to three substituents, such as
chosen from R.sup.11, on the Het ring that is available by chemical
synthesis and is stable is within the scope of this invention.
C.sub.3-7 cycloalkyl refers to an optionally substituted carbocyclic system
of three to seven carbon atoms, which may contain up to two unsaturated
carbon-carbon bonds. Typical of C.sub.3-7 cycloalkyl are cyclopropyl,
cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and
cycloheptyl. Any combination of up to three substituents, such as chosen
from R.sup.11, on the cycloalkyl ring that is available by conventional
chemical synthesis and is stable, is within the scope of this invention.
Any accessible substituted five-membered ring as referred to herein is any
saturated or unsaturated five-membered ring which (i) has up to three
substituents, such as R or R*, wherein the substituents may be present on
any atom or heteroatom that results in a stable structure, and (ii)
contains up to two heteroatoms selected from the group of N, O and S,
wherein S and N may optionally be oxidized, and (iii) is stable and may be
synthesized by one skilled in the chemical arts in a form fused via two
adjacent ring carbon atoms to a phenyl, pyridyl, pyrazinyl, pyridazinyl or
pyrimidinyl ring. Typical of accessible five-membered rings are the common
saturated and unsaturated rings of cyclopentane, cyclopentene, furan,
thiophene, imidazole, pyrrole, thiazole, and oxazole.
Any accessible substituted six-membered ring as referred to herein is an
unsaturated (e.g. aromatic) six-membered ring which (i) has one to three
substituents, such as chosen from R.sup.6, (ii) optionally contains up to
two nitrogens, (iii) is fused via two adjacent carbon atoms to any
accessible substituted five-membered ring, and (iv) is stable and may be
prepared by one skilled in the chemical arts. Typical of accessible
six-membered rings are phenyl, pyridyl, pyrazinyl, pyridazinyl or
pyrimidinyl ring. Representative bicyclic rings formed by the combination
of the accessible six- and five-membered rings are: indene, isoindene,
benzofuran, isobenzofuran, benzothiophene, isobenzothiophene, indole,
indolenine, isobenzazole, 1,5-pyrindine, isoindazole, indoxazine,
benzoxazole, anthranil, benzothiazole, and purine. Phenyl is a preferred
accessible six-membered ring, and imidazole and pyrrole are preferred
accessible five-membered rings. Thus, the preferred bicyclic rings formed
by the combination of the accessible six- and five-membered rings are
benzimidazole and indole.
It will be understood that, with respect to A.sup.1 -A.sup.3, CR.sup.1
R.sup.1' --CR.sup.3 R.sup.3' and NR.sup.1 -NR.sup.3 are saturated sp.sup.3
carbon and nitrogen atoms, respectively, which are singly bonded to the
adjacent ring atoms; CR.sup.1 R.sup.1' --CR.sup.3 R.sup.3' may also
represent an sp.sup.2 carbon atom. It will be further understood that,
with respect to A.sup.1 -A.sup.3, CR.sup.1 --CR.sup.3 and N, they may
represent an unsaturated sp.sup.2 carbon or nitrogen atom, which may be
connected by an endocyclic double bond to an adjacent atom in the ring,
provided such arrangement results in the creation of a stable compound.
N as used herein indicates a nitrogen heterocycle, which may be a saturated
or an unsaturated stable five-, six- or seven-membered monocyclic ring,
containing up to three nitrogen atoms or containing one nitrogen atom and
a heteroatom chosen from oxygen and sulfur, and which may be substituted
on any atom that results in a stable structure, and wherein the nitrogen
heteroatom may be optionally quaternized. Representative of N are
pyrroline, pyrrolidine, imidazole, imidazoline, imidazolidine, pyrazole,
pyrazoline, pyrazolidine, piperidine, piperazine, morpholine, pyridine,
tetrahydropyridine, tetrahydro- and hexahydro-azepine. In particular, N
may be pyrolidinyl, piperidinyl, tetrahydropyridinyl, or piperidinyl.
AA as referred to herein is an amino acid with its carboxyl group
optionally protected, wherein the amino acid may be any of the natural
amino acids or penicillamine. The unprotected carboxyl group is a free
carboxylic acid group. Protecting groups for the carboxyl are esters or
amides which are formed, for instance, when the OH of the carboxy group is
replaced by R.sup.8.
C(O) indicates a carbon doubly bonded to oxygen (eg. carbonyl), C(S)
indicates a carbon doubly bonded to sulfur (eg. thiocarbonyl).
t-Bu refers to the tertiary butyl radical, Boc refers to the
t-butyloxycarbonyl radical, Fmoc refers to the fluorenylmethoxycarbonyl
radical, Ph refers to the phenyl radical, Cbz refers to the
benzyloxycarbonyl radical, BrZ refers to the o-bromobenzyloxycarbonyl
radical, ClZ refers to the o-chlorobenzyloxycarbonyl radical, Bzl refers
to the benzyl radical, 4-MBzl refers to the 4-methyl benzyl radical, Me
refers to methyl, Et refers to ethyl, Ac refers to acetyl, Alk refers to
C.sub.1-4 alkyl, Nph refers to 1- or 2-naphthyl and cHex refers to
cyclohexyl MeArg is N.sup..alpha. -methyl arginine.
DCC refers to dicyclohexylcarbodiimide, DMAP refers to
dimethylaminopyridine, DIEA refers to diisopropylethyl amine, EDC refers
to N-ethyl-N'(dimethylaminopropyl)carbodiimide. HOBt refers to
1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DIEA refers to
diisopropylethylamine, DMF refers to dimethyl formamide, NBS refers to
N-bromo-succinimide, Pd/C refers to a palladium on carbon catalyst, PPA
refers to 1-propanephosphonic acid cyclic anhydride, DPPA refers to
diphenylphosphoryl azide, BOP refers to
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate,
HF refers to hydrofluoric acid, TEA refers to triethylamine, TFA refers to
trifluoroacetic acid, PCC refers to pyridinium chlorochromate.
The compounds of formula (I) are generally prepared by reacting a compound
of the formula (IX) with a compound of the formula (X):
##STR8##
wherein D.sup.1 -D.sup.4 and A.sup.1 -A.sup.3, R and R* are as defined in
formula (I), with any reactive functional groups protected;
L.sup.1 and L.sup.1 are functional groups which are capable of reacting to
form the linkage --(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s --V; and
R.sup.6" is W--(CR'.sub.2).sub.q --Z-- and any portion of the group
--(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s --V-- which is connected to
L.sup.2, with any reactive functional groups protected;
and thereafter removing any protecting groups, and optionally forming a
pharmaceutically acceptable salt.
It will be apparent that the precise identity of L.sup.1 and L.sup.2 will
be dependent upon the site of the linkage being formed. General methods
for preparing the linkage --(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.r
--V-- are describe& for example, in EP-A 0 372 486 and EP-A 0 381 033 and
EP-A 0 478 363, which are incorporated herein by reference.
For instance, if V is CONH, L.sup.1 may be --NH.sub.2, L.sup.2 may be OH
(as in an acid) or Cl (as in an acid chloride), and R.sup.6" may be
W--(CR'.sub.2).sub.q --Z--(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s
--C(O), with any functional groups optionally protected. For example,
R.sup.6" may be (benzyloxycarbonyl-amidino)benzoyl- or (N.sup..alpha.
-Boc,N.sup.guan -Tos)arginyl-. When L.sup.2 is OH, a coupling agent is
used.
Similarly, if V is NHCO, L.sup.1 may be --CO.sub.2 H or CO--Cl, L.sup.2 may
be --NH.sub.2, and R.sup.6" may be W--(CR'.sub.2).sub.q
--Z--(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s --. For example, R.sup.6"
may be (benzyloxycarbonyl-amidino)phenyl,
(benzyloxycarbonylamino)methylbenzyl- or 6-(benzyloxycarbonylamino)hexyl-.
When V is NHSO.sub.2, L.sup.1 may be SO.sub.2 Cl, L.sup.2 may be --NH.sub.2
and R.sup.6" may be as above. When V is SO.sub.2 NH, L.sup.1 may be
--NH.sub.2 and L.sup.2 may be SO.sub.2 Cl. Methods to prepare such
sulfonyl chlorides are disclosed, for instance, in J. Org. Chem., 23, 1257
(1958).
If V is CH.dbd.CH, L.sup.1 may be --CHO, L.sup.2 may be CH.dbd.P--Ph.sub.3
and R.sup.6" may be W(CR'.sub.2).sub.q --Z--(CR'R.sup.10).sub.r
--U--(CR'.sub.2).sub.s --.
Where V is CH.sub.2 CH.sub.2 may be obtained by reduction of a suitably
protected compound wherein V is CH.dbd.CH.
When V is CH.sub.2 O, CH.sub.2 N or, L.sup.1 may be --OH, --NH or --,
respectively; L.sup.2 may be --Br; and R.sup.6" may be
W--(CR'.sub.2).sub.q --Z--(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s --.
For example, R.sup.6" may be (benzyloxycarbonylamino)-methylbenzyl- or
2-(N-benzyl-4-piperidinyl)ethyl. Similarly when U or V is OCH.sub.2,
NR'CH.sub.2 or, L.sup.1 may be --CH.sub.2 Br, and L.sup.2 may be --OH,
--NH or --, respectively.
Compounds wherein V is CHOHCH.sub.2 may be prepared from a suitably
protected compound where V is CH.dbd.CH by the procedure disclosed in J.
Org. Chem., 54, 1354 (1989).
Compounds wherein V is CH.sub.2 CHOH may be obtained from a suitably
protected compound where V is CH.dbd.CH by hydroboration and basic
oxidation as disclosed in Tet. Lett., 31, 231 (1990).
The compounds of formula (IX), wherein A.sup.1 and A.sup.3 are nitrogen and
the five-membered ting is unsaturated, are benzimidazoles and are prepared
by the general methods illustrated by Schemes I-III. The compounds of
formula (IX), wherein A' is carbon and A.sup.3 is nitrogen and the
five-membered ring is unsaturated, are indoles. These compounds are
prepared by the procedure illustrated in Scheme IV. The compounds of
Formula (IX), wherein A.sup.1 is nitrogen and A.sup.3 is oxygen or sulfur
and the five-membered ring is unsaturated, are benzoxazoles and
benzothiazoles, respectively, and are prepared by the methods detailed in
Nestor, et al., J. Med. Chem., 27:320-325 (1984)). Conversion of these
benzoxazoles and benzothiazoles to Formula (I) compounds is carried out
using the methods described hereinbelow for the preparation of Formula (I)
benzimidazoles and indoles. In the Schemes R.sup.1" -R.sup.7" indicate
R.sup.1 -R.sup.7 or a suitable precursor thereof, wherein any functional
groups are protected as known in the art.
##STR9##
Scheme I provides a method of preparing compounds wherein A.sup.1 and
A.sup.3 are nitrogen and A.sup.2 is CR.sup.2. Generally, the synthesis is
begun with a substituted 1,2-diaminobenzene compound. For compounds
wherein (CR'R.sup.10).sub.r --U--(CR.sub.2).sub.s --V-- is CONR'-- the
diaminobenzene starting material is substituted further by a nitro group.
Reaction of this compound with an appropriately substituted
isothiocyanate, such as methyl isothiocyanatopropionate or methyl
isothiocyanatoacetate, or an appropriately substituted carboxylic acid
compound, such as mono-methyl succinate, yields the fused 6-5
benzimidazole ring system with the R.sup.2 -substituent introduced.
Reduction of the nitro group, such as with hydrogen in the presence of a
catalyst, for example, palladium or carbon, in an acetic medium, for
example in acetic acid/methanol (1:1), results in the formation of the
corresponding amino compound. Condensation of the amino group with a
carboxylic acid of the formula, W--(CR'.sub.2).sub.q --Z--CO.sub.2 H, such
as with 4-(Cbz-aminoiminomethyl)benzoic acid or 4-(Boc-aminomethyl)benzoic
acid, in the presence of an amide-forming agent, such as in the presence
of N-ethyl-N'(dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole,
and a base, such as diisopropylethyl amine, in a suitable solvent, such as
in dimethylformamide. Protective groups, such as those for amino or
carboxyl groups, are selectively removed by methods known in the art. For
example, a Cbz-group on a nitrogen atom may be removed by hydrogenation in
the presence of a catalyst, such as palladium on carbon, in an acidic
medium, such as in acetic acid/methanol, and a Boc-group on a nitrogen
atom and/or an ester group on a carboxylic acid may be removed by acid,
for example refluxing aqueous acetic acid.
##STR10##
Scheme II provides a method of preparing compounds wherein A.sup.1 and
A.sup.3 are nitrogen, A.sup.2 is CR.sup.2 and (CR.sup.1 R.sup.10).sub.r
--U--(CR'.sub.2).sub.s --V-- is NR'CO--. According to this scheme, a
1,2-diaminobenzene compound substituted by a carboxylic acid is reacted
with an appropriately substituted isothiocyanate to give R.sup.2
-substituted benzimidazole compounds. The carboxylic acid group is then
converted to the corresponding acid chloride using, for example, refluxing
thionyl chloride and then the acid chloride is reacted with an amine of
the formula, W--(CR'.sub.2).sub.q --Z--NH.sub.2, such as
4-(Cbz-aminoiminomethyl)aniline, in the presence of a base, such as
triethylamine, in an appropriate solvent, such as methylene chloride and
dimethylformamide. The amino and carboxyl protecting groups are removed by
methods known in the art and by methods as described hereinbefore.
##STR11##
Scheme III provides a method of preparing compounds wherein A.sup.1 is N,
A.sup.2 is CR.sup.2, A.sup.3 is NR.sup.3 and
(CR'R.sup.10)--U--(R'.sub.2).sub.s --V-- is CONR'--. According to this
scheme, the Scheme I, Formula 2, benzimidazole compounds are reacted with
an R.sup.3 -substituted halide, such as benzyl bromide, in the presence of
a base, such as sodium hydride, in a suitable solvent, such as
dimethylformamide. These tri-substituted benzimidazoles are then convened
to Formula (D compounds as detailed in Scheme I, Formulae 2 to 4.
##STR12##
Scheme IV provides a method of preparing compounds wherein A.sup.1 is
carbon, A.sup.2 is CR.sup.2, A.sup.3 is nitrogen, and (CR'R.sup.10).sub.r
--U--(CR'.sub.2).sub.s --V-- is NR'CO--. According to this scheme, the
nitro group of a nitrobenzoate ester, such as benzyl 4-nitrobenzoate, is
converted to the corresponding hydroxyamino group using, for example,
hydrazine hydrate in the presence of a reducing agent, such as wet rhodium
on carbon, in a suitable solvent, such as tetrahydrofuran. Reaction of the
hydroxyamino group with an R.sup.2 -substituted acyl Meldrum's acid
(Meldrum's acid is 2,2-dimethyl-1,3-dioxane-4,6-dione.) in an appropriate
solvent, such as acetonitrile, yields N-acetoacetylation of the N-phenyl
hydroxylamine intermediate.. The R.sup.2 -substituted-carboxyindole is
prepared from this intermediate using, for example,
2,2'-azobisisobutyronitrile (AIBN), in an appropriate deoxygenated
solvent, such as xylene. The ester protecting group on the indole
carboxylic acid group is removed by conventional methods, such as, when a
benzyl ester is present, by hydrogenation in the presence of a suitable
catalyst, such as palladium on carbon. Formula (I) compounds are prepared
as detailed in Scheme II, formulae 6 to 8.
Coupling reagents as used herein denote reagents which may be used to form
peptide bonds. Typical coupling methods employ carbodiimides, activated
anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA,
PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are
typical.
Coupling methods to form peptide bonds are generally well known to the art.
The methods of peptide synthesis generally set forth by Bodansky et al.,
THE PRACTICE OF PEPTIDE SYNTHESIS, Springer-Verlag, Berlin, 1984, Ali et
al. in J. Med. Chem., 29, 984 (1986) and J. Med. Chem., 30, 2291 (1987)
are generally illustrative of the technique and are incorporated herein by
reference.
Solution synthesis for the formation of amide or peptide bonds is
accomplished using conventional methods used to form amide bonds.
Typically, the amine or aniline is coupled via its free amino group to an
appropriate carboxylic acis substrate using a suitable carbodiimide
coupling agent, such as N,N' dicyclohexyl carbodiimide (DCC), optionally
in the presence of catalysts such as 1-hydroxybenzotriazole (HOBO and
dimethylamino pyridine (DMAP). Other methods, such as the formation of
activated esters, anhydrides or acid halides, of the free carboxyl of a
suitably protected acid substrate, and subsequent reaction with the free
amine of a suitably protected amine, optionally in the presence of a base,
are also suitable. For example, a protected Boc-amino acid or Cbz-amidino
benzoic acid is treated in an anhydrous solvent, such as methylene
chloride or tetrahydrofuran(THF), in the presence of a base, such as
N-methyl morpholine, DMAP or a trialkylamine, with isobutyl chloroformate
to form the "activated anhydride", which is subsequently reacted with the
free amine of a second protected amino acid or aniline.
Compounds of formula (X) are prepared by conventional methods known in the
art from commercially available materials. W is a generally a basic
functional group attached to Z, optionally via an alkyl chain, and is
protected during the synthesis of R.sup.6 or is introduced into the
molecule after the --(CR'R.sup.10).sub.r --U--(CR'.sub.2).sub.s --V--
linkage has been formed. For example, compounds of formula (x) or formula
(I) wherein W is a suitably substituted R'R"N--, R"R'NC(.dbd.NR'),
R'.sub.2 N(R.sup.13)C.dbd.N--, R"N.dbd.(R.sup.13)C--NR'--, R'.sub.2
N--(R.sub.2 N)C.dbd.N-- or R"R'N(R'N.dbd.)C--NR', are prepared by
conventional methods including those disclosed in EP-A 0 372 486, EP-A 0
381 033 or EP-A 0 478 363, which are incorporated herein by reference.
Compounds of formula (X) wherein W is N are prepared, inter alia, by
methods disclosed in EP-A 0 478 363.
Compounds W is R'.sub.2 N(R'.sub.2 N)C.dbd.N--X-- or
R"R'N.dbd.)C--NR'--X--, and X is O are prepared, inter alia, by methods
disclosed in J. Org. Chem., 51, 5047 (1986).
Compounds wherein W is R'.sub.2 N(R'.sub.2 N)C--N--X-- or
R"R'N(R'N.dbd.)C--NR'--X--, and X is N.dbd.CR', are prepared, inter alia,
by methods disclosed in United States Pat. No. 3,714,253 and Eur. J. Med.
Chem.-Chim. Ther., 20, 25 (1985).
Compounds wherein W is R'.sub.2 N(R'.sub.2 N)C.dbd.N--X-- or
R"R'N(R'N.dbd.)C--NR'--X--, and X is C(O), are prepared, inter alia, by
methods disclosed in U.S. Pat. No. 3,714,253 and Can. J. Chem., 43, 3103
(1965).
Compounds wherein W is R'.sub.2 NR'C(.dbd.NR')-- may be prepared, inter
alia, by methods disclosed in J. Het. Chem., 16, 1063 (1979) or J. Her.
Chem., 26, 125 (1989).
Compounds wherein W is R'.sub.2 NR'NC(--NR')-- are prepared by conventional
methods including those disclosed in Syn., 583 (1974).
Compounds wherein W is R'R"NR'N-- are prepared, inter alia, by methods
disclosed in J. Prakt. Chem., 36, 29 (1967).
Compounds wherein W is R'R"NR'NCO-- are prepared, inter alia, by methods
disclosed in Bull. Chem. Soc. Jpn., 43, 2257 (1970).
Compounds wherein W is R"R'NC(.dbd.NR')Y, and Y is S, are prepared, inter
alia, by methods disclosed in Chem. Lett., 1379 (1986).
Compounds of formula (X) or formula (I), wherein W is R"R'NC(.dbd.NR')Y and
Y is O, are prepared by conventional methods including those disclosed in
Japanese Patent 2022751.
The reactive functional groups of the sidechains of each synthetic fragment
are suitably protected as known in the art. Suitable protective groups are
disclosed in Green, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, John Wiley and
Sons, New York, 1981. For example, the Boc, Cbz, phthaloyl or Fmoc group
may be used for protection of an amino or amidino group. The Boc group is
generally preferred for protection of an .alpha.-amino group. A t-Bu, cHex
or benzyl ester may be used for the protection of the side chain carboxyl.
A benzyl group or suitably substituted benzyl group (eg. 4-methoxy-benzyl
or 2,4-dimethoxy-benzyl) is used to protect the mercapto group or the
hydroxyl group. The tosyl group may be used for protection of the
imidazolyl group and tosyl or nitro group for protection of the guanidino
group. A suitably substituted carbobenzyloxy group or benzyl group may be
also be used for the hydroxyl group or amino group. Suitable substitution
of the carbobenzyloxy or benzyl protecting groups is ortho and/or para
substitution with chloro, bromo, nitro or methyl, and is used to modify
the reactivity of the protective group. Except for the Boc group, the
protective groups for the amino moiety are, most conveniently, those which
are not removed by mild acid treatment. These protective groups are
removed by such methods as catalytic hydrogenation, sodium in liquid
ammonia or I-IF treatment, as known in the art.
Modification of amino groups especially on the six-membered ring of the
bicyclic system, may be accomplished by alkylation, sulfonylation,
cyanation or acylation as is generally known in the art.
Acid addition salts of the compounds of formula (I) are prepared in a
standard manner in a suitable solvent from the parent compound and an
excess of an acid, such as hydrochloric, hydrobromic, sulfuric,
phosphoric, acetic, maleic, succinic or methanesulfonic. The acetate salt
form is especially useful. Certain of the compounds form inner salts or
zwitterions which may be acceptable. Cationic salts are prepared by
treating the parent compound with an excess of an alkaline reagent, such
as a hydroxide, carbonate or alkoxide, containing the appropriate cation;
or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++,
Mg++ and NH.sub.4 + are specific examples of cations present in
pharmaceutically acceptable salts.
This invention provides a pharmaceutical composition which comprises a
compound according to formula (I) and a pharmaceutically acceptable
carrier. Accordingly, the compounds of formula (I) may be used in the
manufacture of a medicament. Pharmaceutical compositions of the compounds
of formula (I) prepared as hereinbefore described may be formulated as
solutions or lyophilized powders for parenteral administration. Powders
may be reconstituted by addition of a suitable diluent or other
pharmaceutically acceptable carrier prior to use. The liquid formulation
may be a buffered, isotonic, aqueous solution. Examples of suitable
diluents are normal isotonic saline solution, standard 5% dextrose in
water or buffered sodium or ammonium acetate solution. Such formulation is
especially suitable for parenteral administration, but may also be used
for oral administration or contained in a metered dose inhaler or
nebulizer for insufflation. It may be desirable to add excipients such as
polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene
glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in a
emulsion or syrup for oral administration. Pharmaceutically acceptable
solid or liquid carriers may be added to enhance or stabilize the
composition, or to facilitate preparation of the composition. Solid
carriers include starch, lactose, calcium sulfate dihydrate, terra alba,
magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
Liquid carriers include syrup, peanut oil, olive oil, saline and water.
The carrier may also include a sustained release material such as glyceryl
monostearate or glyceryl distearate, alone or with a wax. The amount of
solid carrier varies but, preferably, will be between about 20 mg to about
1 g per dosage unit. The pharmaceutical preparations are made following
the conventional techniques of pharmacy involving milling, mixing,
granulating, and compressing, when necessary, for tablet forms; or
milling, mixing and filling for hard gelatin capsule forms. When a liquid
carrier is used, the preparation will be in the form of a syrup, elixir,
emulsion or an aqueous or non-aqueous suspension. Such a liquid
formulation may be administered directly p.o. or filled into a soft
gelatin capsule.
For rectal administration, the compounds of this invention may also be
combined with excipients such as cocoa butter, glycerin, gelatin or
polyethylene glycols and molded into a suppository.
This invention also provides a method of inhibiting platelet aggregation
and clot formation in a mammal, especially a human, which comprises the
internal administration of a peptide of formula (I) and a pharmaceutically
acceptable carrier. Indications for such therapy include acute myocardial
infarction (AMI), deep vein thrombosis, pulmonary embolism, dissecting
anurysm, transient ischemia attack (TIA), stroke and other infarct-related
disorders, and unstable angina. Chronic or acute states of
hyper-aggregability, such as disseminated intravascular coagulation (DIC),
septicemia, surgical or infectious shock, post-operative and post-partum
trauma, cardiopulmonary bypass surgery, incompatible blood transfusion,
abruptio placenta, thrombotic thrombocytopenic purpura (TTP), snake venom
and immune diseases, are likely to be responsive to such treatment In
addition, the compounds of this invention may be useful in a method for
the prevention of metastatic conditions, the prevention or treatment of
fungal or bacterial infection, inducing immunostimulation, and the
prevention or treatment of diseases in which bone resorption is a factor.
The compounds of this invention are administered either orally or
parenterally to the patient, in a manner such that the concentration of
drug in the plasma is sufficient to inhibit platelet aggregation, or other
such indication. The pharmaceutical compositions containing the compounds
of this invention are administered at a dose between about 0.2 to about 50
mg/kg in a manner consistent with the condition of the patient. For acute
therapy, parenteral administration is preferred. For persistent states of
hyperaggregability, an intravenous infusion of the compound in 5% dextrose
in water or normal saline is most effective, although an intramuscular
bolus injection may be sufficient.
For chronic, but noncritical, states of platelet aggregability, oral
administration of a capsule or tablet, or a bolus intramuscular injection
is suitable. The compounds of this invention are administered one to four
times daily at a level of about 0.4 to about 50 mg/kg to achieve a total
daily dose of about 0.4 to about 200 mg/kg/day.
This invention further provides a method for inhibiting the reocclusion of
an mew or vein following fibrinolytic therapy, which comprises internal
administration of a compound of formula (I) and a fibrinolytic agent. It
has been found that administration of certain compounds in fibrinolytic
therapy either prevents reocclusion completely or prolongs the time to
reocclusion.
When used in the context of this invention the term fibrinolytic agent is
intended to mean any compound, whether a natural or synthetic product,
which directly or indirectly causes the lysis of a fibrin clot.
Plasminogen activators are a well known group of fibrinolytic agents.
Useful plasminogen activators include, for example, anistreplase,
urokinase (UK), pro-urokinase (pUK), streptokinase (SK), tissue
plasminogen activator (tPA) and routants, or variants, thereof, which
retain plasminogen activator activity, such as variants which have been
chemically modified or in which one or more amino acids have been added,
deleted or substituted or in which one or more or functional domains have
been added, deleted or altered such as by combining the active site of one
plasminogen activator with the fibrin binding domain of another
plasminogen activator or fibrin binding molecule. Other illustrative
variants include tPA molecules in which one or more glycosylation sites
have been altered. Preferred among plasminogen activators are variants of
tPA in which the primary amino acid sequence has been altered in the
growth factor domain so as to increase the serum half-life of the
plasminogen activator. tPA Growth factor variants are disclosed, e.g., by
Robinson et al., EP-A 0 297 589 and Browne et al., EP-A 0 240 334. Other
variants include hybrid proteins, such as those disclosed in EP 0 028 489,
EP 0 155 387 and EP 0 297 882, all of which are incorporated herein by
reference. Anistreplase is a preferred hybrid protein for use in this
invention. Fibrinolytic agents may be isolated from natural sources, but
are commonly produced by traditional methods of genetic engineering.
Useful formulations of tPA, SK, UK and pUK are disclosed, for example, in
EP-A 0 211 592, EP-A 0 092 182 and U.S. Pat. No. 4,568,543, all of which
are incorporated herein by reference. Typically the fibrinolytic agent may
be formulated in an aqueous, buffered, isotonic solution, such as sodium
or ammonium acetate or adipate buffered at pH 3.5 to 5.5. Additional
excipients such as polyvinyl pyrrolidone, gelatin, hydroxy cellulose,
acacia, polyethylene, glycol, mannitol and sodium chloride may also be
added. Such a composition can be lyophilized.
The pharmaceutical composition may be formulated with both the compound of
formula (I) and fibrinolytic in the same container, but formulation in
different containers is preferred. When both agents are provided in
solution form they can be contained in an infusion/injection system for
simultaneous administration or in a tandem arrangement.
Indications for such therapy include myocardial infarction, deep vein
thrombosis, pulmonary embolism, stroke and other infarct-related
disorders. The compound of this invention is administered just prior to,
at the same time as, or just after patenteral administration of tPA or
other fibrinolytic agent. It may prove desirable to continue treatment
with the claimed compounds for a period of time well after reperfusion has
been established to maximally inhibit post-therapy reocclusion. The
effective dose of tPA, SK, UK or pUK may be from 0.5 to 5 mg/kg and the
effective dose of the peptide may be from about 0.1 to 25 mg/kg.
For convenient administration of the inhibitor and the fibrinolytic agent
at the same or different times, a kit is prepared, comprising, in a single
container, such as a box, carton or other container, individual bottles,
bags, vials or other containers each having an effective mount of the
inhibitor for parenteral administration, as described above, and an
effective amount of tPA, or other fibrinolytic agent, for parenteral
administration, as described above. Such kit can comprise, for example,
both pharmaceutical agents in separate containers or the same container,
optionally as lyophilized plugs, and containers of solutions for
reconstitution. A variation of this is to include the solution for
reconstitution and the lyophilized plug in two chambers of a single
container, which can be caused to admix prior to use. With such an
arrangement, the fibrinolytic and the compound of this invention may be
packaged separately, as in two containers, or lyophilized together as a
powder and provided in a single container.
When both agents are provided in solution form, they can be contained in an
infusion/injection system for simultaneous administration or in a tandem
arrangement. For example, the platelet aggregation inhibitor may be in an
i.v. injectable form, or infusion bag linked in series, via tubing, to the
fibrinolytic agent in a second infusion bag. Using such a system, a
patient can receive an initial bolus-type injection or infusion of the
inhibitor followed by an infusion of the fibrinolytic agent.
The pharmacological activity of the compounds of this invention is assessed
by their ability to inhibit the binding of 3H-SK&F 107260, a known
RGD-fibrinogen antagonist, to the GPIIbIIIa receptor, their ability to
inhibit platelet aggregation, in vitro, and their ability to inhibit
thrombus formation in vivo.
Inhibition of RGD-mediated GPIIb-IIIa Binding
Purification of GPIIb-IIIa
Ten units of outdated, washed human platelets (obtained from Red Cross)
were lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCl, pH
7.4, 140 mM NACl, 2 mM CaCl.sub.2 at 4.degree. C. for 2 h. The lysate was
centrifuged at 100,000 g for 1 h. The supernatant obtained was applied to
a 5 mL lentil lectin sepharose 4B column (E. Y. Labs) preequilibrated with
20 mM Tris-HCl, pH 7.4, 100 mM NACl, 2 mM CaCl.sub.2, 1% octylglucoside
(buffer A). After 2 h incubation, the column was washed with 50 mL cold
buffer A. The lectin-retained GPIIb-IIIa was eluted with buffer A
containing 10% dextrose. All procedures were performed at 4.degree. C. The
GPIIb-IIIa obtained was >95% pure as shown by SDS polyacrylamide gel
electrophoresis.
Incorporation of GPIIb-IIIa in Liposomes
A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%) (Avanti
Polar Lipids) were dried to the walls of a glass tube under a stream of
nitrogen. Purified GPIIb-IIIa was diluted to a final concentration of 0.5
mg/mL and mixed with the phospholipids in a protein:phospholipid ratio of
1:3 (w:w). The mixture was resuspended and sonicated in a bath sonicator
for 5 min. The mixture was then dialyzed overnight using 12,000-14,000
molecular weight cutoff dialysis tubing against a 1000-fold excess of 50
mM Tris-HCl, pH 7.4, 100 mM NACl, 2 mM CaCl.sub.2 (with 2 changes). The
GPIIb-IIIa-containing liposomes wee centrifuged at 12,000 g for 15 min and
resuspended in the dialysis buffer at a final protein concentration of
approximately 1 mg/mL. The liposomes were stored at -70.degree. C. until
needed.
Competitive Binding to GPIIb-IIIa
The binding to the fibrinogen receptor (GPIIb-IIIa) was assayed by an
indirect competitive binding method using ›.sup.3 H!-SK&F-107260 as an
RGD-type ligand. The binding assay was performed in a 96-well filtration
plate assembly (Millipore Corporation, Bedford, Mass.) using 0.22 um
hydrophilic durapore membranes. The wells were precoated with 0.2 mL of 10
.mu.g/mL polylysine (Sigma Chemical Co., St. Louis, Mo.) at room
temperature for 1 h to block nonspecific binding. Various concentrations
of unlabeled benzadiazapines were added to the wells in quadruplicate.
›.sup.3 H!-SK&F-107260 was applied to each well at a final concentration
of 4.5 nM, followed by the addition of 1 .mu.g of the purified platelet
GPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h at
room temperature. The GPIIb-IIIa-bound ›.sup.3 H!-SK&F-107260 was
seperated from the unbound by filtration using a Millipore filtration
manifold, followed by washing with ice-cold buffer (2 times, each 0.2 mL).
Bound radioactivity remaining on the filters was counted in 1.5 mL Ready
Solve (Beckman Instruments, Fullerton, Calif.) in a Beckman Liquid
Scintillation Counter (Model LS6800), with 40% efficiency. Nonspecific
binding was determined in the presence of 2 .mu.M unlabeled SK&F-107260
and was consistently less than 0.14% of the total radioactivity added to
the samples. All data points are the mean of quadruplicate determinations.
The compounds of this invention inhibit ›.sup.3 H!-SK&F 107260 binding
with Ki in the range of about 40 nM to about 50 .mu.M. Preferred compounds
have Ki of less than 100 nM.
Inhibition of Platelet Aggregation
Blood was collected (citrated to prevent coagulation) from, naive, adult
mongrel dogs. Platelet rich plasma, PRP, was prepared by centrifugation at
150.times.g for 10 min at room temperature. Washed platelets were prepared
by centrifuging PRP at 800.times.g for 10 min. The cell pellet thus
obtained was washed twice in Tyrode's buffer (pH 6.5) without Ca.sup.++
and resuspended in Tyrode's buffer (pH 7.4) containing 1.8 mM Ca.sup.++ at
3.times.10.sup.5 cells/ml. Compounds were added 3 min prior to the agonist
in all assays of platelet aggregation. Final agonist concentrations were
0.1 unit/ml thrombin and 2 mM ADP (Sigma). Aggregation was monitored in a
Chrono-Log Lumi-Aggregometer. Light transittance 5 min after addition of
the agonist was used to calculate percent aggregation according to the
formula % aggregation=›(90-CR)O(90-10)!.times.100, where CR is the chart
reading, 90 is the baseline, and 10 is the PRP blank reading. IC50's were
determined by plotting ›% inhibition of aggregation! vs. ›concentration of
compound!. Compounds were assayed at 200 mM and diluted sequentially by a
factor of 2 to establish a suitable dose response curve.
The compounds of this invention inhibit the aggregation of human platelets
stimulated with ADP with IC50 of about 0.45 to about 50 .mu.M. Preferred
compounds have IC50 of less than 1 .mu.M.
To assess the stability of the compounds to plasma proteases, the compounds
were incubated for 3 h (rather than 3 min) in the PRP prior to addition of
the agonist.
In Vivo Inhibition of Platelet Aggregation
In vivo inhibition of thrombus formation is demonstrated by recording the
systemic and hemodynamic effects of infusion of the peptides into
anesthetized dogs according to the methods described in Aiken et al.,
Prostaglandins, 19, 629 (1980).
The examples which follow are intended to in no way limit the scope of this
invention, but are provided to illustrate how to make and use the
compounds of this invention. Many other embodiments will be readily
apparent and available to those skilled in the art.
EXAMPLES
Example 1
›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-acetic Acid
(i) Methyl 2-amino-N-acetate 5-nitrobenzimidazole
Under argon, 4-nitro-1,2-phenylenediamine (7.15 g, 46.7 mmol) and methyl
2-isothiocyanatoacetate (6.12 g, 46.7 mmol) were mixed in dimethylformide
(20 mL) and toluene (100 mL). The reaction was stirred at 65.degree. C.
for 45 minutes before cooling down to room temperature and adding mercuric
oxide (10.13 g, 46.7 mmol) in one portion. The reaction was stirred at
65.degree. C. overnight to give a dark brown solution. The reaction was
filtered and rinsed with hot methanol, concentrated in vacuo to give a
brownish-red oil. Flash chromatography (methanol/methylene chloride, 1:9)
afforded the desired product (11.25 g, 96%).
(ii) Methyl 2-amino-N-acetate-5-aminobenzimidazole, acetate salt
The compound of Example 1(i) (4.04 g, 16.14) was dissolved in methanol
acetic acid (50:50 mL) and then 10% palladium on carbon (1.5 g) was added.
The reaction was hydrogenated at 45 psi for 2.5 hours, filtered through
Celite.RTM., rinsed with methanol, and concentrated under vacuo to give
the desired product (3.65 g, 81%), which was used without further
purification.
(iii)
›5-(4-Cbz-aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-acetate,
methyl ester
The compound of Example 1(ii) (0.52 g, 1.86 mmol) was mixed with
4-(Cbz-aminoiminomethyl)benzoic acid (0.55 g, 1.86 mmol),
1-hydroxybenzotriazole (0.3 g 2.22 mmol), and
N-ethyl-N'(dimethylaminopropyl)carbodiimide (0.43 g, 2.21 mmol) in
dimethylformamide (12 mL). The reaction mixture was cooled down to
0.degree. C. before adding diisopropylethylamine (0.74 g, 5.74 mmol)
slowly, and then it was stirred at room temperature under argon overnight.
The dark brown reaction mixture was poured into a flask containing 50 mL
of ice and 5% sodium bicarbonate (6.8 mL) to give a white precipitate
which was filtered and washed with cold water to give, upon drying, the
desired product (0.68 g, 73%) that was used without further purification.
(iv) ›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-acetate
methyl ester, acetate salt
Starting with the compound of Example 1(iii), the title compound was
prepared following the procedure for the preparation of the compound of
Example 1(ii).
(v) ›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-acetic
acid, trifluoroacetic acid salt
The compound of Example 1(iv) (0.25 g, 0.12 mmol) was mixed in 10% acetic
acid (12 mL) and stirred at 110.degree. C. overnight. The solvent was
removed under vacuo to give the crude product (0. 11 g, 94%). Purification
on reverse phase prep HPLC (CH.sub.3 CN:H.sub.2 O, 0.1% TFA) afforded the
analytically pure desired product. ESMS: m/e 353.2 (M+H).sup.+, 351.0
(M-H).sup.-. Anal. Calcd. for C.sub.17 H.sub.16 N.sub.6 O.sub.3.2C.sub.2
HF.sub.3 O.sub.2.1/2H.sub.2 O; C,42.79; H, 3.25; N, 14.26. Found: C,
42.84; H, 3.04; N, 14.00.
Example 2
›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-propionic Acid
(i) Methyl 3-isothiocyanatopropionate
To .beta.-alanine methyl esterhydrochloric acid salt suspended in methylene
chloride (60 mL) was added triethylamine (10.87 g, 107.4 mmol). The
reaction mixture was cooled downed to -15.degree. C. before adding carbon
disulfide (4.1 g, 53.8 mmol) dissolved in chloromethane (30 mL) dropwise
over 25 minutes. The reaction was warmed to 10.degree. C. and stirred for
10 minutes. before lowering the temperature to 0.degree. C. and adding
ethyl chloroformate (5.83 g, 53.7 mmol) dissolved in chloromethane (5 mL)
dropwise over 15 minutes. The temperature was increased to room
temperature and stirred for 20 minutes before lowering to 0.degree. C.
Triethylamine (5.43 g, 53.7 mmol) was then added. The temperature was
increased to room temperature and then stirred for 30 minutes. The
reaction mixture was washed with water (50 mL), 1N hydrochloric acid (50
mL), 5% sodium bicarbonate and water (50 mL), dried over magnesium
sulfate, filtered and concentrated under vacuo to give 7.3 g. of crude
product. Flash chromatography (ethyl acetate/hexane, 1:4) afforded the
desired product (4.03 g, 52%).
(ii) Methyl 2-amino-N-propanoate-5-nitrobenzimidazole
Starting with the compound of Example 2(i), the title compound was prepared
following the procedure of Example 1(i).
(iii) Methyl 2-amino-N-propanoate 5-aminobenzimidazole, acetate salt
Starting with the compound of Example 2(ii), the title compound was
prepared following the procedure of Example 1(ii).
(iv)
›5-(4-Cbz-aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-propanoate
, methyl ester
Starting with the compound of Example 2(iii), the title compound was
prepared following the procedure of Example 1(iii).
(v) ›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-propanoate,
methyl ester, acetate salt
Starting with the compound of Example 2(iv), the title compound was
prepared following the procedure of Example 1(ii).
(vi) ›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-amino-N-propanoic
acid, trifluoroacetic acid salt
Starting with the compound of Example 2(v), the title compound was prepared
following the procedure of Example 1(v). ESMS: m/e 367.2 (M+H).sup.+.
Anal. Calcd. for C.sub.18 H.sub.18 N.sub.6 O.sub.3.2C.sub.2 HF.sub.3
O.sub.2.1/2H.sub.2 O: C, 43.79; H, 3.51; N, 13.93. Found: C, 43.61; H,
3.67; N, 14.01.
Example 3
›5-(4-Aminoiminomethyl)phenylaminocarbonyl!benzimidazole-2-amino-N-acetic
Acid
(i) Methyl 2-amino-N-acetate-5-carboxybenzimidazole
Following the procedure of Example 1(i) and starting with
3,4-diaminobenzoic acid in place of 4-nitro-1,2-phenylenediamine, the
title compound was prepared.
(ii) 4-(Cbz-aminoiminomethyl)analine
To 4-(aminoiminomethyl)analine dihydrochloride (4.0 g, 19.2 mmol) suspended
in tetrahydrofuran (100 mL) and water (20 mL) was added slowly 5N sodium
hydroxide (11.6 mL) at 0.degree. C. After 2 minutes, Cbz--Cl was added
(3.28 g, 19.2 mmol) dropwise. The reaction was stirred at 0.degree. C. for
1 hour before removing most of the solvent in vacuo. The residue was
dissolved in methylene chloride (700 mL), washed with 100 mL of water,
dried over sodium sulfate, filtered, and concentrated in vacuo to give the
desired product (4.5 g, 87%). This crude product was used without further
purification.
(iii)
›5-(4-Cbz-aminoiminomethyl)phenylaminocarbonyl!benzimidazole-2-amino-N-ace
tate, methyl ester
The compound of Example 3(i) (0.87 g, 3.49 mmol) was converted to the acid
chloride by refluxing with thionyl chloride (7 mL) overnight. The excess
thionyl chloride was removed and the residue was mixed with the compound
of Example 3(ii) (1.0 g, 3.7 mmol) in methylene chloride (12 mL) and
dimethylformamide (5 mL). Triethylamine (1.13 g, 11.2 mmol) was added at
0.degree. C., and then the reaction was stirred at room temperature
overnight under argon. The reaction mixture was poured into a flask
containing 50 mL of ice to give a brown precipitate (1.76 g, 94%). Flash
chromatography (methanol/methylene chloride, 7:93) afforded the desired
product.
(iv)
›5-(4-Aminoiminomethyl)phenylaminocarbonyl!benzimidazole-2-amino-N-acetate
, methyl ester, acetate salt
Starting with the compound of Example 3(iii), the title compound was
prepared following the procedure of Example 1(ii).
(v)
›5-(4-Aminoiminomethyl)phenylaminocarbonyl!benzimidazole-2-amino-N-acetic
acid, trifluoroacetic acid salt
Starting with the compound of Example 3(iv), the title compound was
prepared following the procedure of Example 1(v). ESMS: m/e 353.2
(M+H).sup.+, 351.2 (M-H).sup.-. Anal. Calcd. for C.sub.17 H.sub.16 N.sub.6
O.sub.3.2C.sub.2 HF.sub.3 O.sub.2 : C, 43.46; H, 3.13; N, 14.48. Found: C,
43.76; H, 3.56; N, 14.07.
Example 4
›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic Acid
(i) Methyl 2-propanoate 5-nitrobenzimidazole
4-Nitro 1,2-phenylenediamine (1.54 g, 10 mmol) and mono methyl succinate
(2.0 g, 15.1 mmol) were mixed in 4N hydrochloric acid (30 mL) to give a
greenish-brown solution. The reaction was stirred at 110.degree. C. for 24
hours, and the solvent was removed in vacuo to give a purple solid. Flash
chromatography (methanol/methylene chloride, 5:95) afforded the desired
product (0.5 g, 20%).
(ii) Methyl 2-propanoate-5-aminobenzimidazole, acetate salt
Starting with the compound of Example 4(i), the title compound was prepared
following the procedure of Example 1(ii).
(iii) ›5-(4-Cbz-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoate,
methyl ester
Starting with the compound of Example 4(ii), the title compound was
prepared following the procedure of Example 1(iii).
(iv) ›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-propanoate methyl
ester, acetate salt
Starting with the compound of Example 4(iii), the title compound was
prepared following the procedure of Example 1(ii).
(v) ›5-(4-Aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic acid,
trifluoroacetic acid salt
Starting with the compound of Example 4(iv), the title compound was
prepared following the procedure of Example 1(v). ESMS: m/e 353.2
(M+H).sup.+, 350.0 (M-H).sup.-. Anal. Calcd. for C.sub.18 H.sub.17 N.sub.5
O.sub.3.2.33C.sub.2 HF.sub.3 O.sub.2 : C, 44.11; H, 3.16; N, 11.35. Found:
C, 44.51; H, 3.28; N, 10.99.
Example 5
›5-(4-Aminomethyl)benzoylamino!benzimidazole-2-amino-N-acetic Acid
(i) ›5-(4-Boc-aminomethyl)benzoylamino!benzimidazole-2-amino-N-acetate,
methyl ester
Starting with the compound of Example 1(ii) (0.7 g, 2.5 mmol) and using
4-(Boc-aminomethyl)benzoic acid (2.5 g, 0.63 mmol), the title compound was
prepared following the procedure of Example 1(iii).
(ii) ›5-(4-Aminomethyl)benzoylamino!benzimidazole-2-N-amino-acetic acid,
trifluoroacetic acid salt
Starting with the compound of Example 5(i), the rifle compound was prepared
following the procedure of Example 1(v). ESMS: m/e 340.2 (M+H).sup.+,
338.2 (M-H).sup.-. Anal. Calcd. for C.sub.17 H.sub.17 N.sub.5
O.sub.3.2.5C.sub.2 HF.sub.3 O.sub.2 : C,42.31; H, 3.15; N, 11.22. Found:
C, 42.09; H, 3.24; N, 11.31.
Example 6
1-N-Benzyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
Acid (i) 1-N-Benzyl-5-nitrobenzimidazole-2-propanoate, methyl ester
The compound of Example 4(i) (1.0 g, 4.0 mmol) and sodium hydride (0.11 g,
4.4 mmol) were mixed with tetrahydrofuran (30 mL) and dimethylformamide (5
mL) at room temperature to give a clear brown solution. Benzyl bromide
(0.76 g, 4.4 mmol) was added in one portion and then the reaction was
stirred overnight at room temperature. The reaction mixture was
partitioned in methylene chloride:water (250:50 mL). The methylene
chloride extract was dried over sodium sulfate, filtered, and concentrated
in vacuo to give a brownish oil. Flash chromatagraphy (ethyl
acetate/hexane, 1:1) separated the two regio-isomers. The higher R.sub.f
spot was shown to be the title compound (6(i)(a), 0.68, 50%); whereas, the
lower R.sub.f spot was the other isomer, 1-N-Benzyl
6-nitrobenzimidazole-2-propanoate methyl ester (6(i)(b), 0.30 g, 22%).
(ii) 1-N-Benzyl-5-aminobenzimidazole-2-propanoate methyl ester, acetate
salt
Starting with the compound of Example 6(i)(a), the title compound was
prepared following the procedure of Example 1(ii).
(iii)
1-N-Benzyl-›5-(4-Cbz-aminoiminomethyl)benzoylamino!benzimidazole-2-propano
ate, methyl ester
Starting with the compound of Example 6(ii), the title compound was
prepared following the procedure of Example 1(iii).
(iv)
1-N-Benzyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoate,
methyl ester, acetate salt
Starting with the compound of Example 6(iii), the title compound was
prepared following the procedure of Example 1(ii).
(v)
1-N-Benzyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic a
cid, trifluoroacetic acid salt
Starting with the compound of Example 6(iv), the title compound was
prepared following the procedure of Example 1(v). ESMS: m/e 442.2
(M+H).sup.+. Anal. Calcd. for C.sub.25 H.sub.23 N.sub.5 O.sub.3.2.5C.sub.2
HF.sub.3 O.sub.2.2H.sub.2 O: C, 47.25; H, 3.90; N, 9.18. Found: C, 47.59;
H, 4.18; N, 8.84.
Example 7
1-N-Benzyl-›6-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
Acid
(i) 1-N-Benzyl-6-aminobenzimidazole-2-propanoate methyl ester, aceate salt
Starting with the compound of Example 6(i)(b), the title compound was
prepared following the procedure of Example 1(ii).
(ii)
1-N-Benzyl-›6-(4-Cbz-aminoiminomethyl)benzoylamino!benzimidazole-2-propano
ate, methyl ester
Starting with the compound of Example 7(i), the title compound was prepared
following the procedure of Example 1(iii).
(iii)
1-N-Benzyl-›6-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoate,
methyl ester, acetate salt
Starting with the compound of Example 7(ii), the title compound was
prepared following the procedure of Example 1(ii).
(iv)
1-N-Benzyl-›6-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
acid, trifluoroacetic acid salt
Starting with the compound of Example 7(iii), the title compound was
prepared following the procedure of Example 1(v). ESMS: m/e 442.2
(M+H).sup.+, 440.0 (M-H).sup.-. Anal. Calcd. for C.sub.25 H.sub.23 N.sub.5
O.sub.3.2.5C.sub.2 HF.sub.3 O.sub.2.1.5H.sub.2 O: C, 47.82; H, 3.81; N,
9.29. Found: C, 48.14; H, 3.84; N, 9.20.
Example 8
1-N-Phenethyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoic
Acid
(i) 1-N-Phenethyl-5-nitrobenzimidazole-2-propanoate, methyl ester
Following the procedure for the preparation of the compounds of Example
6(i)(a) and (b), the two regio-isomers were prepared using phenethyl
bromide in place of benzyl bromide. The higher R.sub.f spot was shown to
be the title compound; whereas, the lower R.sub.f spot was the other
isomer, 1-N-phenethyl 6-nitrobenzimidazole-2-propanoate, methyl ester.
(ii) 1-N-Phenethyl 5-aminobenzimidazole-2-propanoate, methyl ester, acetate
salt
Starting with the compound of Example 8(i), the title compound was prepared
following the procedure of Example 1(ii).
(iii)
1-N-Phenethyl-›5-(4-Cbz-aminoiminomethyl)benzoylamino!benzimidazole-2-prop
anoate, methyl ester
Starting with the compound of Example 8(ii), the title compound was
prepared following the procedure of Example 1(iii).
(iv)
1-N-Phenethyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoa
te, methyl ester, acetate salt
Starting with the compound of Example 8(iii), the title compound was
prepared following the procedure of Example 1(ii).
(v)
1-N-Phenethyl-›5-(4-aminoiminomethyl)benzoylamino!benzimidazole-2-propanoi
c acid, trifluoroacetic acid salt
Starting with the compound of Example 8(iv), the title compound was
prepared following the procedure of Example 1(v). ESMS: m/e 456.2
(M+H).sup.+, 454.2 (M-H).sup.-. Anal. Calcd. for C.sub.26 H.sub.25 N.sub.5
O.sub.3.2.5C.sub.2 HF.sub.3 O.sub.2.2H.sub.2 O: C, 47.94; H, 4.08; N,
9.01. Found: C, 47.58; H, 4.15; N, 8.87.
Example 9
5-›4-Aminoiminomethyl)phenylaminocarbonyl!indole-2-propanoic Acid
(i) Benzyl 4-(hydroxyamino)benzoate
Hydrazine hydrate (containing 55% hydrazine, 1.5 mL, 26.25 mmol, 1.05
equiv.) was added dropwise over 1 minute to a mixture containing benzyl
4-nitrobenzoate (Brewster, J. H.; Ciotti, C. J., Jr. J. Am. Chem. Soc.
1955, 77, 6214; 6.43 g, 25 mmole, 1 equiv.), 5% rhodium on carbon (wet,
625 mg), and tetrahydrofuran (50 ml) at 0.degree. C. under argon. When the
addition was complete, the mixture was warmed to room temperature, stirred
for 4 hours, and then was filtered through Celite.RTM.. Concentration of
the filtrate gave crude benzyl 4-(hydroxyamino)benzoate as a yellow oil
which was used without further purification. Pure benzyl
4-(hydroxyamino)benzoate, obtained from a separate preparation by silica
gel chromatography (30% ethyl acetate/toluene) was used for
characterization purposes: TLC(30% ethyl acetate/toluene) R.sub.f 0.52;
.sup.1 H NMR (250 MHz, CDCl.sub.3) .delta. 7.99 (d, J=8.6 Hz, 2 H),
7.26-7.55 (m, 5 H), 6.97 (d, J=8.6 Hz, 2H), 5.33 (s, 2H); IR (CHCl.sub.3)
3550, 3315, 1703, 1606, 1273, 1169, 1101, 693 cm.sup.-1 ; MS(DC/NH.sub.3)
261.2 (M+NH.sub.4).sup.+, 245.2 (M+MH.sub.4 -16).sup.+, 228.1
(M+H-16).sup.+.
(ii)
N-›4-(Benzyloxycarbonyl)phenyl!-N-hydroxy-5-(methoxycarbonyl)-3-oxopentana
mide
A solution of benzyl 4-(hydroxyamino)benzoate (25 mmol, 1 equiv.) and
3-methoxycarbonylpropionyl Meldrum's Acid (Oikawa, Y.; Sugano, K.;
Yonemitsu, O. J. Org. Chem. 1978, 43, 2087; 7.75 g, 30 mmol, 1.2 equiv.)
in dry acetonitrile (125 mL) was heated at reflux under argon. After 45
minutes, the solvent was removed in vacuo and the residue was
chromatographed rapidly on silica gel (30% ethyl acetate/toluene). The
title compound (7.99 g, 80%, impure) was obtained as a yellow solid. Pure
material was used for characterization purposes: TLC (30% ethyl
acetate/toluene) R.sub.f 0.39; .sup.1 H NMR (250 MHz, CDCl.sub.3) .delta.
8.03 (app. d, 2 H), 7.68 (app. d, 2 H), 7.28-7.54 (m, 5 H), 5.63 (s, 1 H),
5.34 (s, 2 H), 3.73 (s, 3 H), 3.06 (d, J=16.7 Hz, 1 H), 2.89 (d, J=16.7
Hz, 1 H), 2.52-2.82 (m, 2 H), 2.25-2.60 (m, 2 H); IR (CHCl.sub.3)
3100-3500 (br), 1710, 1357, 1272, 1174, 1108 cm.sup.-1 ; MS (DCI/NH.sub.3)
417.2 (M+NH.sub.4).sup.+, 401.2 (M+NH.sub.4 -16)+, 400 (M+H)+, 384.1
(M+H-16).sup.+, 287.1, 245, 228.1, 148.1.
(iii) Methyl 5-benzyloxycarbonylindole-2-propanoic acid
A mixture of the compound of Example 1(ii), (7.99 g, 20 mmol, 1 equiv.) and
AIBN (65.7 mg, 0.4 mmol, 0.02 equiv.) in p-xylene (Aldrich HPLC grade, 100
mL) was deoxygenated through a single evacuation/argon flush cycle, and
the resulting mixture was heated to reflux under argon. A yellow solution
was produced which steadily darkened as heating was continued. After 0.5
hour, the reaction was concentrated and the residue was chromatographed on
silica gel (twice: run 1: 40% ethyl acetate/hexane; run 2: 2:1
hexane/ethyl acetate. Some crystallization on the column occurred both
times). The title compound (1.67 g, 25%; 20% from benzyl 4-nitrobenzoate)
was obtained as a yellow solid. TLC (40% ethyl acetate/hexane) R.sub.f
0.51; .sup.1 H NMR (400MHz, CDCl.sub.3) .delta. 8.82(brs, 1 H), 8.32 (s, 1
H), 7.88 (dd, J=8.6, 1.3 Hz, 1 H), 7.28-7.50 (m, 6 H), 6.31 (s, 1 H), 5.38
(s, 2 H), 3.73 (s, 3 H), 3.07 (t, J=6.5 Hz, 2 H), 2.75 (t, J=6.5 Hz, 2 H);
IR (CHCl.sub.3) 3200-3500 (br), 1720 (shoulder), 1700, 1303, 1255, 1232
cm.sup.-1 ; MS (ESMS) 338.0 (M+H).sup.+.
(iv) Methyl 5-carboxyindole-2-propanoic acid
10% Palladium on carbon (212.8 mg, 0.2 mmol, 0.2 equiv.) was added to a
solution of the compound of Example 9(iii) (338.4 mg, 1 mmol, 1 equiv.) in
1:1 ethyl acetate/methanol (30 mL), and the resulting mixture was stirred
under hydrogen (40 psi). After 1 hour, the reaction was filtered through
Celite.RTM. to remove the catalyst, and the filter pad was washed with
both ethyl acetate and methanol. Concentration of the filtrate gave the
title compound (240.8 mg, 97%) as an off-white solid. TLC (10%
MeOH/CHCl.sub.3) R.sub.f 0.56; .sup.1 H NMR (400 MHz, CDCl.sub.3 plus
DMSO-d.sub.6) .delta. 8.23 (s, 1 H), 7.77 (dd, J=8.5, 1.4 Hz, 1 H), 7.31
(d, J=8.5 Hz, 1 H), 6.27 (s, 1 H), 3.70 (s, 3 H), 3.09 (t, J=7.5 Hz, 2 H),
2.78 (t, J=7.5 Hz, 2 H); MS (ESMS) 248.0 (M+H).sup.+.
(v) ›5-›4-Aminoiminomethyl)benzylaminocarbonyl!indole-2-propanoic acid
The title compound is prepared following the procedure of Example
3(iii)-(v), replacing methyl 2-amino-N-acetate-5-carboxybenzimidazole with
methyl 5-carboxyindole-2-propanoic acid.
Example 10
5-›(4-Piperidinylacetyl)amino!-1H-benzimidazole-2-butanoic Acid
(i) Methyl 2-butanoate 5-nitrobenzimidazole
Starting with 4-Nitro 1,2-phenylenediamine (10.0 g, 65.3 mmol) and mono
methyl glutarate (15.5 g, 106 mmol), the title compound was prepared
following the procedure of Example 4(i).
(ii) Methyl 2-butanoate 5-aminobenzimidazole
Starting with the compound of Example 10(i), the title compound was
prepared following the procedure of Example 1(ii).
(iii) 5-›(4-N-Boc-piperidinylacetyl)amino!-1H-benzimidazole-2-butanoate
methyl ester
Starting with the compound of Example 10(ii) (0.35 g, 1.5 mmol) and using
4-N-Boc-piperidinylacetic acid (0.40 g, 1.65 mmol), the title compound was
prepared following the procedure of Example 1(iii).
(iv) 5-›(4-Piperidinylacetyl)amino!-1H-benzimidazole-2-butanoic acid,
hydrochloride salt
The compound of Example 10(iii) (0.35 g, 0.76 mmol) was dissolved in 10%
acetic acid (25 mL) and stirred at 115.degree. C. for 16 hours before
adding 1.0N hydrochloric acid (5 mL) directly into the reaction mixture
and stirring for additional one hour. The solvent was removed under vacuo
to give the crude product (0.33 g, 94%). Purification on reverse phase
prep high pressure liquid chromatography (acetonitrile: water, 0.1%
trifluoroacetic acid) afforded the trifluoroacetic acid salt. The
analytically pure product was converted to hydrochloride salt by
dissolving in 1.0N hydrochloric acid and removing the solvent via
lyophilization. ESMS: m/e 345.2 (M+H).sup.+, 343.2 (M-H).sup.-. Anal.
Calcd. for C.sub.18 H.sub.24 N.sub.4 O.sub.3.3HCl.2H.sub.2 O: C, 44.14; H,
6.38; N, 11.44. Found: C, 44.51; H, 6.34; N, 11.69.
Example 11
5-›(4-Piperidinylacetylamino!-1H-benzimidazole-2-propanoic Acid
(i) 5-›(4-N-Boc-piperidinylacetyl)amino!-1H-benzimidazole-2-propanoate,
methyl ester
Starting with the compound of Example 4(ii) (0.136 g, 0.62 mmol) and using
4-N-Boc-piperidinylacetic acid (0.166 g, 0.68 mmol), the title compound
was prepared following the procedure of Example 1(iii).
(ii) 5-›(4-Piperidinylacetyl)amino!-1H-benzimidazole-2-propanoic acid,
hydrochloride salt
Starting with the compound of Example 11(i), the title compound was
prepared following the procedure of Example 10(iv). ESMS :m/e 331
(M+H).sup.+. Anal. Calcd. for C.sub.17 H.sub.22 N.sub.4 O.sub.3.2HCl. TFA.
2.75H.sub.2 O; C, 40.04; H, 6.03; N, 10.99. Found: C, 40.13; H, 6.29; N,
10.95.
Example 12
5-›(4-Piperidinylcarbonyl)amino!-1H-benzimidazole-2-butanoic Acid
(i) 5-›(4-N-Boc-piperidinylcarbonyl)amino!-1H-benzimidazole-2-butanoate,
methyl ester
Starting with the compound of Example 10(ii) (0.35 g, 1.5 mmol) and using
4-N-Boc-hexahydroisonicotinic acid (0.38 g, 1.65 mmol), the title compound
was prepared following the procedure of Example 1(iii).
(ii) 5-›(4-Piperidinylcarbonyl)amino!-1H-benzimidazole-2-butanoic acid,
hydrochloride salt
Starting with the compound of Example 12(i), the tide compound was prepared
following the procedure of Example 10(iv). ESMS :m/e 331.2 (M+H).sup.+.
Anal. Calcd. for C.sub.17 H.sub.22 N.sub.4 O.sub.4.2HCl.2H.sub.2 O: C,
46.48; H, 6.42; N, 12.75. Found: C, 46.31; H, 6.11; N, 12.59.
Example 13
5-››4-(Aminoiminomethyl)benzoyl!amino!-1H-benzimidazole-2-acetic Acid
(i) Ethyl 5-nitrobenzimidazole-2-acetate
Concentrated nitric acid (5 mL) was added to concentrated sulfonic acid (5
mL) at 0.degree. C., and the solution was cooled thoroughly for an
additional 10-15 minutes. Ethyl benzimidazole-2-acetate (Buchi, J.;
Zwicky, H.; Aebi, A., Archiv. Der Pharmazie 1960, 293, 758; 1.09 g, 5.34
mmol, 1 equiv.) was added portionwise over several minutes The resulting
yellow solution was stirred at 0.degree. C. for 20 minutes then was poured
carefully onto cracked ice (100 g). The pH was adjusted to 5 with cold 20%
sodium carbonate (50-75 mL) and the mixture was extracted with ethyl
acetate (3.times.50 mL). Drying (sodium sulfate), concentration, and
silica gel chromatography (3:2 ethyl acetate/toluene) gave ethyl
5-nitrobenzimidazole-2-acetate (1.23 g, 92%) as a yellow solid. TLC (3:2
ethyl acetate/toluene) R.sub.f 0.44; .sup.1 H NMR (400 MHz, CDCl.sub.3)
.delta. 8.54 (br s, 1H), 8.21 (dd, J=8.9, 2.1 Hz, 1H), 7.58-7.70 (m, 1H),
4.31 (q, J=7.1 Hz, 2 H), 4.14 (s, 2 H), 1.35 (t, J=7.1 Hz, 3 H); IR
(chloroform) 3390, 1727, 1523, 1345, 1309 cm.sup.-1 ; MS (ESMS) 250.0
(M+H).sup.+.
(ii) Ethyl
5-››4-(N-Cbz-aminoiminomethyl)benzoyl!amino!-1H-benzimidazole-2-acetate
(a) Ethyl 5-aminobenzimidazole-2-acetate
A mixture of ethyl 5-nitrobenzimidazole-2-acetate (206 mg, 0.83 mmol, 1
equiv.), 10% palladium on carbon (88 mg, 0.08 mmol, 0.1 equiv.), and
absolute ethanol (8 mL) was stirred at room temperature under hydrogen
(balloon). After 45 minutes, the reaction was filtered through
Celite.RTM., and the filtrate was concentrated to afford crude ethyl
5-aminobenzimidazole-2-acetate. This was used without further
purification.
(b) Ethyl
5-››4-(N-Cbz-aminoiminomethyl)benzoyl!amino!-1H-benzimidazole-2-acetate
N-Ethyl-N'(dimethylaminopropyl)carbodimmide (191 mg, 1.0 mmol, 2 equiv.)
was added all at once to a solution of ethyl
5-aminobenzimidazole-2-acetate (0.83 mmol, 1 equiv.),
N-Cbz-(aminoiminomethyl)benzoic acid (272 mg, 0.91 mmol, 1.1 equiv.),
1-hydroxybenzotriazole hydrate (135 mg, 1.0 mmol, 1.2 equiv.), and
diisopropylethylamine (0.22 mL, 1.25 mmol, 1.5 equiv.) in dry
dimethylformamide (4 mL) at 0.degree. C. under argon. The resulting
solution was warmed to room temperature and stirred overnight, then was
concentrated on the rotavap. The residue was partitioned between water (20
mL) and ethyl acetate (20 mL), and the layers were separated. The aqueous
layer was extracted with ethyl acetate (2.times.20 mL), and all ethyl
acetate layers were combined methanol was added to the combined organic
extracts to dissolve suspended solids, and the resulting solution was
dried (magnesium sulfate) and concentrated to a yellow solid.
Chromatography on silica gel (3:2 acetone/chloroform) gave ethyl
5-››4-(N-Cbz-aminoiminomethyl)benzoyl!amino!-1H-benzimidazole-2-acetate
(316.9 mg, 76% for two steps) as a yellow solid. TLC (3:2
acetone/chloroform) R.sub.f 0.50; .sup.1 H NMR (400 MHz, CD.sub.3 OD)
.delta. 8.08 (s, 1H), 8.04 (d, J=8.5 Hz, 2 H), 7.98 (d, J=8.5 Hz, 2 H),
7.52 (d, J=8.7 Hz, 1H), 7.25-7.50 (m, 6 H), 5.21 (s, 2 H), 4.22 (q, J=7.2
Hz, 2 H), 1.27 (t, J=7.2 Hz, 3 H); The protons .alpha.-to the carbethoxy
group had exchanged with D from the CD.sub.3 OD; MS (ESMS) 500.2
(M+H).sup.+, 366.2.
(iii) 5-››4-(Aminoiminomethyl)benzoyl!amino!-1H-benzimidazole-2-acetic acid
10% Palladium on carbon (68 mg, 0.063 mmol, 0.01 equiv.) was added to a
solution of ethyl
5-››4-(N-Cbz-aminoiminomethyl)benzoyl!amino!-1H-benzimidazole-2-acetate
(316.9 mg, 0.63 mmol, 1 equiv.) and trifluoracetic acid (0.049 mL,, 0.63
mmol, 1 equiv.) in absolute ethanol (12 mL), and the mixture was stirred
at room temperature under hydrogen (balloon). After 1 hour, the mixture
was filtered through Celite.RTM. and the filtrate was concentrated to a
yellow solid.
This solid was dissolved in absolute ethanol (10 mL) and 1.0N sodium
hydroxide (1.9 mL, 1.9 mmol, 3 equiv.), and the resulting yellow solution
was stirred at room temperature. A precipitate soon separated. The mixture
was stirred for 3 hour, then more 1.0N sodium hydroxide (1.3 mL, 1.26
mmol, 2 equiv.) was added. Stirring at room temperature was continued for
another 3 hour, then water (6 mL) was added to afford a clear solution,
which was stirred at room temperature overnight. The reaction was
concentrated to dryness and the residue was diluted with 1:1
acetonitrile/water (6 mL). The mixture was cooled to 0.degree. C., and
trifluoroacetic acid (0.49 mL, 6.3 mmol, 10 equiv.) was added. The
resulting yellow solution was concentrated to dryness and the residue was
purified by reversed-phase flash chromatography on C-18 silica gel (5%
acetonitrile/water/0.1% trifluoracetic acid (300 mL) then 7.5%
acetonitrile/water/0.1% trifluoracetic acid). The product-containing
fractions were combined, concentrated on the rotavap to 20 mL, and
lyophilized to afford
5-››4-(aminoiminomethyl)benzoyl!amino!-1H-benzimidazole-2-acetic acid-2
trifluoroacetic acid salt.multidot.1.75 water (218.3 mg, 58 as a colorless
powder. HPLC (PRP-1 column; 10% CH.sub.3 CN/water/0.1% trifluoracetic
acid) k'=1.10; .sup.1 H NMR (400 MHz, CD.sub.3 OD) .delta. 8.47 (s, 1H),
8.19 (d, J=8.4 Hz, 2 H), 7.96 (d, J=8.4 Hz, 2 H), 7.67-7.77 (m, 2 H); The
protons .alpha.-to the carbethoxy group had exchanged with D from the
CD.sub.3 OD; MS (ESMS) 338.0 (M+H).sup.+, 294.0 (M+H--CO.sub.2).sup.+,
169.4 (M+2 H).sup.+, 146.4; Anal. Calcd for C.sub.17 H.sub.15 N.sub.5
O.sub.3.2 CF.sub.3 CO.sub.2 H-1.75 water:. C, 42.26; H, 3.46; N, 11.73.
Found: C, 42.21; H, 3.14; N, 11.56.
Example 14
Parenteral Dosage Unit Composition
A preparation which contains 20 mg of the compound of Example 4 as a
sterile dry powder is prepared as follows: 20 mg of the compound is
dissolved in 15 ml of distilled water. The solution is filtered under
sterile conditions into a 25 ml multi-dose ampoule and lyophilized. The
powder is reconstituted by addition of 20 ml of 5% dextrose in water (D5W)
for intravenous or intramuscular injection. The dosage is thereby
determined by the injection volume. Subsequent dilution may be made by
addition of a metered volume of this dosage unit to another volume of D5W
for injection, or a metered dose may be added to another mechanism for
dispensing the drug, as in a bottle or bag for IV drip infusion or other
injection-infusion system.
Example 15
Oral Dosage Unit Composition
A capsule for oral administration is prepared by mixing and milling 50 mg
of the compound of Example 4 with 75 mg of lactose and 5 mg of magnesium
stearate. The resulting powder is screened and filled into a hard gelatin
capsule.
Example 16
Oral Dosage Unit Composition
A tablet for oral administration is prepared by mixing and granulating 20
mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of the
compound of Example 4 with a 10% gelatin solution. The wet granules are
screened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg stearic acid;
and compressed into a tablet.
The foregoing is illustrative of the making and using of this invention.
This invention, however, is not limited to the precise embodiments
described herein, but encompasses all modifications within the scope of
the claims which follow.
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